The present invention relates to sweet proteins. Specifically, this invention relates to Brazzein protein that has been modified to improve its sweetness characteristics.
The most widely used natural sweetener, sugar (sucrose), has significant problems associated with its use (especially causing weight gain by users). Many other sweeteners either have undesirable side effects or are deficient in certain respects. For example, aspartame loses its sweetness when exposed to elevated temperatures for long periods. This renders aspartame unsuitable for use in most baking applications.
Moreover, most existing artificial sweeteners have temporal sweetness profiles which do not adequately match that of sugar. For example, their sweetness may die out sooner or leave an undesirable after taste, and/or may be perceived sooner than sugar. It may therefore be desirable to mix an existing artificial sweetener with one or more other sweeteners having different temporal profiles (so as to create a mixed sweetener that more closely matches the overall temporal sweetness profile of sugar).
Only relatively few sweet proteins (as distinguished from sweet carbohydrates) have been found in nature. One protein that appears to be particularly promising is Brazzein (SEQ ID NO: 1). See also U.S. Pat. Nos. 5,346,998 and 5,527,555, PCT publications WO 94/19467 and 95/31547, and J. Caldwell et al., Solution Structure Of The Thermostable Sweet-tasting Protein Brazzein, 5 Nature Structural Biology 427–431 (1998). This protein is particularly desirable because it is stable when subjected to the level of heat typically present during baking of foods. The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
While naturally occurring Brazzein has certain desirable characteristics, there are some concerns regarding its temporal profile and the amount of the sweetener needed for threshold perception. Prior research had not been able to identify any consensus sequence or structure in Brazzein responsible for sweetness or the sweetness profile.
As described in WO 00/61759, attempts were made to improve certain sweetness characteristics of Brazzein through the substitution of Ala or Arg in replacement for an existing amino acid, and/or the addition of Ala or Arg, and/or the truncation of an existing terminal amino acid, of Brazzein. Some of these changes increased sweetness potency, while others decreased it.
Similarly, in H. Izawa et al. Pept. Sci.: Present Future, Proc. Int. Pept. Symp., 1st (1999)(Ed. Y. Shimonishi) there was a description of Ala substitutions for certain amino acids of Brazzein, with some results showing increased sweetness, while others showed decreased sweetness.
While these prior art modified Brazzein proteins are of significant interest, there is still a need for the development of other sweeteners with differing and/or improved sweetness potency and/or temporal characteristics.